Systems and methods for detecting single-molecule binding kinetics

Abstract

Provided herein are methods of detecting single-molecule binding kinetics that include contacting a set of single molecules with a surface of an optically transparent substrate and irradiating the surface of the substrate with light having an incident angle selected to achieve total reflection of the light. In some embodiments, the methods include collecting light scattered by the surface and by the subset of the single molecules having the binding association with the surface of the substrate to form a series of raw image data sets, and detecting multiple individual binding and leaving events for the subset of the single molecules having the binding association with the surface of the substrate over one or more selected durations using the series of raw image data sets to produce a detected individual binding and leaving event data set. Systems, computer readable media, and additional methods are also provided.

What is claimed is: 
     
         1 . A method of detecting single-molecule binding kinetics, the method comprising:
 contacting a set of single molecules with a surface of an optically transparent substrate;   irradiating the surface of the substrate with light having an incident angle selected to achieve total reflection of the light, thereby scattering light from the surface and from at least a subset of the single molecules having a binding association with the surface of the substrate;   collecting light scattered by the surface and by the subset of the single molecules having the binding association with the surface of the substrate to form a series of raw image data sets; and,   detecting multiple individual binding and leaving events for the subset of the single molecules having the binding association with the surface of the substrate over one or more selected durations using the series of raw image data sets to produce a detected individual binding and leaving event data set, thereby detecting the single-molecule binding kinetics.   
     
     
         2 . The method of  claim 1 , comprising determining one or more single molecule binding durations by pairing dynamic information in the detected individual binding and leaving event data set. 
     
     
         3 . The method of  claim 1 , comprising determining at least one association rate (k a ) and at least one dissociation rate (k d ) using the detected individual binding and leaving event data set. 
     
     
         4 . The method of  claim 1 , comprising identifying specific binding events and non-specific binding events in the detected individual binding and leaving event data set. 
     
     
         5 . The method of  claim 4 , comprising filtering identified non-specific binding events from the detected individual binding and leaving event data set. 
     
     
         6 . The method of  claim 1 , comprising determining at least one association rate (k a ) and at least one dissociation rate (k d ), and identifying specific binding events and non-specific binding events using the detected individual binding and leaving event data set in a single step. 
     
     
         7 . The method of  claim 1 , comprising applying at least one data processing protocol to the series of raw image data sets to produce processed image data sets, which data processing protocol is configured to:
 remove noise from the series of raw image data sets;   magnify one or more morphological characteristics of the single molecules in the series of raw image data sets;   select one or more candidate pixels if an intensity level of a given candidate pixel in the series of raw image data sets exceeds a threshold intensity level to provide one or more sets of selected candidate pixels;   extract location and intensity information for the single molecules in one or more selected regions that comprise the selected candidate pixels to provide a set of location and intensity information; and/or,   identify the multiple individual binding and leaving events using the set of location and intensity information.   
     
     
         8 . The method of  claim 7 , comprising removing the noise by row averaging and normalizing the series of raw image data sets. 
     
     
         9 . The method of  claim 7 , comprising applying a Haar-like array to magnify the one or more morphological characteristics of the single molecules in the series of raw image data sets. 
     
     
         10 . The method of  claim 7 , comprising fitting a two-dimensional (2D) model to the one or more selected regions that comprise the selected candidate pixels to extract the set of location and intensity information. 
     
     
         11 . The method of  claim 7 , comprising deleting given candidate pixels in the sets of selected candidate pixels when the given candidate pixels have a bias level that exceeds a predetermined bias threshold level in fitting from at least one Gaussian blob. 
     
     
         12 . A system for detecting single-molecule binding kinetics, the system comprising:
 an optically transparent substrate;   a fluid handler for flowing a set of single molecules over a surface of the substrate;   a light source configured to irradiate the surface with light having an incident angle selected to achieve total reflection of the light;   a camera;   a collection optical system configured to collect light scatted by the surface and by at least a subset of single molecules having a binding association with the surface; and   a controller operably connected at least to the fluid handler, the light source, the camera, and the collection optical system, which controller comprises a processor, and a memory communicatively coupled directly or remotely to the processor, the memory storing non-transitory computer executable instructions which, when executed by the processor, perform operations comprising:   contacting the set of single molecules with the surface of the substrate using the fluid handler;   irradiating the surface of the substrate with light having the incident angle selected to achieve total reflection of the light using the light source to thereby scatter light from the surface and from at least a subset of the single molecules having a binding association with the surface of the substrate;   collecting light scattered by the surface and by the subset of the single molecules having the binding association with the surface of the substrate using the camera and the collection optical system to form a series of raw image data sets; and   detecting multiple individual binding and leaving events for the subset of the single molecules having the binding association with the surface of the substrate over one or more selected durations using the series of raw image data sets to produce a detected individual binding and leaving event data set.   
     
     
         13 . The system of  claim 12 , wherein the collection optical system is configured to collect light scatted by the surface and by the subset of the single molecules having the binding association with the surface of the substrate from the opposite side of the incident and reflected light. 
     
     
         14 . The system of  claim 12 , wherein the collection optical system is configured to collect light scatted by the surface and by the subset of the single molecules having the binding association with the surface of the substrate on the same side of the incident and reflected light, but avoid the collection of the reflected light. 
     
     
         15 . The system of  claim 12 , wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising:
 determining one or more single molecule binding durations by pairing dynamic information in the detected individual binding and leaving event data set.   
     
     
         16 . The system of  claim 12 , wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising:
 determining at least one association rate (k a ) and at least one dissociation rate (k d ) using the detected individual binding and leaving event data set.   
     
     
         17 . The system of  claim 12 , wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising:
 identifying specific binding events and non-specific binding events in the detected individual binding and leaving event data set.   
     
     
         18 . The system of  claim 12 , wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising:
 determining at least one association rate (k a ) and at least one dissociation rate (k d ), and identifying specific binding events and non-specific binding events using the detected individual binding and leaving event data set in a single step.   
     
     
         19 . The system of  claim 12 , wherein the non-transitory computer executable instructions which, when executed by the processor, further perform operations comprising:
 applying at least one data processing protocol to the series of raw image data sets to produce processed image data sets, which data processing protocol is configured to:   remove noise from the series of raw image data sets;   magnify one or more morphological characteristics of the single molecules in the series of raw image data sets;   select one or more candidate pixels if an intensity level of a given candidate pixel in the series of raw image data sets exceeds a threshold intensity level to provide one or more sets of selected candidate pixels;   extract location and intensity information for the single molecules in one or more selected regions that comprise the selected candidate pixels to provide a set of location and intensity information; and/or,   identify the multiple individual binding and leaving events using the set of location and intensity information.   
     
     
         20 . A computer readable media, comprising non-transitory computer executable instructions which, when executed by a processor, perform operations comprising:
 contacting a set of single molecules with a surface of a substrate using a fluid handler;   irradiating the surface of the substrate with light having the incident angle selected to achieve total reflection of the light using a light source to thereby scatter light from the surface and from at least a subset of the single molecules having a binding association with the surface of the substrate;   collecting light scattered by the surface and by the subset of the single molecules having the binding association with the surface of the substrate using a camera and a collection optical system to form a series of raw image data sets; and   detecting multiple individual binding and leaving events for the subset of the single molecules having the binding association with the surface of the substrate over one or more selected durations using the series of raw image data sets to produce a detected individual binding and leaving event data set.